Construction of magnetic nanochains to achieve magnetic energy coupling in scaffold

Background Fe3O4 nanoparticles are highly desired for constructing endogenous magnetic microenvironment in scaffold to accelerate bone regeneration due to their superior magnetism. However, their random arrangement easily leads to mutual consumption of magnetic poles, thereby weakening the magnetic stimulation effect. Methods In this study, magnetic nanochains are synthesized by magnetic-field-guided interface co-assembly of Fe3O4 nanoparticles. In detail, multiple Fe3O4 nanoparticles are aligned along the direction of magnetic force lines and are connected in series to form nanochain structures under an external magnetic field. Subsequently, the nanochain structures are covered and fixed by depositing a thin layer of silica (SiO2), and consequently forming linear magnetic nanochains (Fe3O4@SiO2). The Fe3O4@SiO2 nanochains are then incorporated into poly l-lactic acid (PLLA) scaffold prepared by selective laser sintering technology. Results The results show that the Fe3O4@SiO2 nanochains with unique core–shell structure are successfully constructed. Meanwhile, the orderly assembly of nanoparticles in the Fe3O4@SiO2 nanochains enable to form magnetic energy coupling and obtain a highly magnetic micro-field. The in vitro tests indicate that the PLLA/Fe3O4@SiO2 scaffolds exhibit superior capacity in enhancing cell activity, improving osteogenesis-related gene expressions, and inducing cell mineralization compared with PLLA and PLLA/Fe3O4 scaffolds. Conclusion In short, the Fe3O4@SiO2 nanochains endow scaffolds with good magnetism and cytocompatibility, which have great potential in accelerating bone repair.


Introduction
Recent scaffolds lack the capacity to effectively modulate cell growth or tissue reconstruction, resulting in slow bone regeneration and even failure of bone implantation [1][2][3][4]. It is well known that cells are magnetically sensitive due to the diamagnetism of cell membranes [5], and exposure to magnetic fields served to alter membrane flux and regulate ion channels and biochemical pathways [6,7]. In this case, a series of cell behaviors will be mediated. Inspired by this, it is significant to construct an endogenous magnetic microenvironment in bone scaffold to mediate cell behaviors and tissue regeneration via magnetic stimulation.
As a typical magnetic material, Fe 3 O 4 nanoparticles have been widely used in tissue engineering field due to their superior magnetic properties and good biocompatibility [8]. In addition, Fe 3 O 4 nanoparticles enable to decompose into oxygen and iron in the body and can be easily removed from the body after degradation by oxygen transport and metabolism [9]. In our previous study [10], the introduction of Fe 3 O 4 nanoparticles into bone scaffolds constructed an endogenous magnetic microenvironment that enhanced cell activity and accelerated new bone generation. It is worth noting that the random arrangement of Fe 3 O 4 nanoparticles in scaffold easily leads to mutual repulsive of magnetic poles between adjacent nanoparticles, thereby weakening the magnetic strength. In this case, the scaffold cannot fully exert its regulation on cell behaviors.
The directional assembly of Fe 3 O 4 nanoparticles into ordered nanochain structures is expected to solve the above problem. Under an external magnetic field, the internal magnetic dipole moment of Fe 3 O 4 nanoparticles enable to be rapidly deflected to the direction of magnetic field [11,12]. Moreover, the attractive magnetic dipole interaction will drive multiple Fe 3 O 4 nanoparticles to assemble into ordered magnetic nanochain structures along with magnetic force lines [13,14]. Compared with randomly arranged Fe 3 O 4 nanoparticles, the magnetic nanochain structures can realize the magnetic energy coupling between the nanoparticles, thereby enhancing the magnetic strength [15][16][17]. Li et al. synthesized uniform linear cobalt nanochains with a coating layer of polyvinylpyrrolidone under an external magnetic field [18]. Wan et al. synthesized magnetic nanochains and fixated with a protective mesoporous silicon shell for osteoclasttargeted inhibition and heterogeneous nanocatalysis [19].
In this study, Fe 3 O 4 @SiO 2 magnetic nanochains with core-shell structure were synthesized utilizing magneticfield-guided interface co-assembly of nanoparticles. In short, Fe 3 O 4 nanoparticles were firstly coated with a layer of protective SiO 2 film. Then, the above products were aligned and assembled into nanochains under an external magnetic field. Thereafter, the nanochains were fixated by further deposition of SiO 2 shell to permanently preserve their structure. Then, the Fe 3 O 4 @SiO 2 nanochains were loaded into PLLA and prepared into porous PLLA/ Fe 3 O 4 @SiO 2 scaffold using selective laser sintering (SLS). The microscopic morphology, chemical composition, and physicochemical properties of Fe 3 O 4 @SiO 2 were analyzed. The cell activity, osteogenic differentiation and mineralization abilities induced by the PLLA/Fe 3 O 4 @ SiO 2 scaffold were investigated and analyzed in detail. Moreover, the osteogenesis-related gene expressions of runt-related transcription factor-2 (Runx2), osteopontin (OPN), osteocalcin (OCN) and osterix (OSX) in cells cocultured with the scaffold were assessed.

Synthesis of one-dimensional Fe3O4@SiO2 nanochains
The Fe 3 O 4 @SiO 2 nanochains were prepared through the magnetic-field-guided interface co-assembly of Fe 3 O 4 nanoparticles, as shown in Fig. 1. First, 20 mg of Fe3O4 nanoparticles was dispersed in 60 mL ethanol-aqueous solution by ultrasonication for 20 min. Subsequently, 5 mL of ammonia solution (28 wt.%) was dropped into the resulting solution with mechanical agitating (800 rpm) for 30 min. Then, the agitating speed was reduced to 300 rpm, and 10 mL of ethanol solution with TEOS (V ethanol :V TEOS = 9:1) were dropped in the solution to coat Fe 3 O 4 nanoparticles with a layer of protective SiO 2 film in the early stage of the sol-gel reaction of TEOS. After reaction for 15 min, the above solution was exposed in a static magnetic field (55 mT-65 mT) for 2 min to induce the linear arrangement of Fe 3 O 4 nanoparticles without stirring, forming numerous nanochain structures. After standing for another 10 min, the nanochain structures were further fixated with an addition SiO 2 shell. Finally, the fixated core-shell Fe 3 O 4 @SiO 2 magnetic nanochains were magnetically separated from the suspension, washed three times with ethanol and distilled water and dried in a vacuum oven. The representative PLLA/Fe 3 O 4 @SiO 2 scaffold with honeycomb structure was fabricated via SLS technology. In detail, the PLLA/Fe 3 O 4 @SiO 2 powders were paved on the powder bed and selectively laser scanned according to the designed three-dimensional model, with laser power at 2.5 W, scanning speed at 100 mm/s and scanning distance at 0.24 mm [20,21]. The modeling platform gradually descended with each layer of powders was sintered until the scaffold was completely formed.

Measurement and characterization
The morphologies of the Fe 3 O 4 @SiO 2 nanochains were observed by transmission electron microscope (TEM, TALOS F200X, USA). The chemical structure, compositions and crystal structures of the Fe 3 O 4 @SiO 2 or/and mL. All the cells were incubated in a humidified condition with 5% CO 2 at 37 °C.
The cells at a density of 4 × 10 3 cells/well were incubated on the surface of sterilized scaffolds in 48-well plates containing DMEM, in which the DMEM was updated daily. After incubating for 3 and 7 days, each cell-scaffold sample was washed with PBS three times, and then immersed in 5% glutaraldehyde for 30 min to fix cells. Thereafter, the cell-scaffold samples were dehydrated with 30%, 50%, 70%, 80%, 90%, 95% and absolute ethyl alcohol in sequence and dried for 12 h at room temperature. After coated with gold, the cell morphology on each scaffold was observed by scanning electron microscope (SEM).
Cell viabilities on the scaffolds were studied by a live/ dead staining kit (Beyotime, China). At the specified time (3 and 7 days), the culture medium was removed, and the cells were detached from scaffolds and rinsed using PBS three times. Subsequently, the cells were stained with 2 μM Calcein-AM for 30 min at 37 °C. Finally, the stained cells were visualized using a fluorescence microscope (Olympus, Japan).
Cell proliferation was quantitatively assessed using Cell Counting Kit-8 (CCK-8, Beyotime, China) assay. The cellscaffold samples were harvested from culture medium after 1, 4 and 7 days of cultivating. Then, the samples were washed with PBS and transferred into 96-well plates containing 100 μL CCK-8 reagent. After incubating for 2 h, the absorbance of the solution was detected utilizing a microplate reader (Thermal, USA) at 450 nm. Each group was carried out three parallel experiments.
The alkaline phosphatase (ALP) activity of the cells on scaffolds was determined to assess the osteogenic differentiation. After incubating for 7 days, the harvested cells from samples were washed with PBS 3 times and fixed with 4% paraformaldehyde for 15 min. Then, ALP staining kit (Beyotime, China) was dropped to stain the cells, and the stained cells were monitored using an inverted microscope (TE2000U, Japan).
The mineralization nodules of MG-63 cells cocultured with scaffolds were qualitatively investigated using Alizarin Red staining. The cells were seeded on scaffolds in 6-well plates for 7 days at a density of 1 × 10 4 cells/mL. After that, the cell-scaffold samples were fixed using 4% paraformaldehyde and rinsed with PBS. Subsequently, the samples were stained with 0.04 M Alizarin Red for 10 min. After rinsing, the samples were observed under a light microscope.
Scaffold-mediated cell differentiation and osteogenesis were further studied. For this, the expression of several relative genes containing runt-related transcription factor-2 (Runx2), osteopontin (OPN), osteocalcin (OCN) and osterix (OSX) were detected with quantitative real-time polymerase chain reactions (RT-PCR). After incubating for 3 and 7 days, the RNA isolation of cells was employed using TRIzox reagent, and then the RNA was reverse transcribed to cDNA using PrimeScript 1st strand cDNA synthesis kit. Finally, the levels of Runx2, OPN, OCN and OSX were calculated using the 2 −ΔΔCt method. Each sample was analyzed three times.

Statistical analysis
All data were conducted by Student's t-test for independent samples and presented as means ± standard deviation, where **p < 0.01 and *p < 0.05 represented significant difference.

Fe3O4@SiO2 nanochains
The representative one-dimensional structure of the Fe 3 O 4 @SiO 2 nanochains were observed using TEM. As shown in Fig. 2a and b, the Fe 3 O 4 @SiO 2 nanochains presented a unique core-shell structure, in which Fe 3 O 4 nanoparticles served as cores with a diameter of about 100 nm and SiO 2 layers served as shells with a thickness of about 25 nm. Based on Fast Fourier transform (FFT, Fig. 2c and d), the interplanar distance between adjacent lattice fringes was 0.25 nm, which was in great agreement with the (311) lattice planes of Fe 3 O 4 . Moreover, the diffraction rings depicted in the selected area electron diffraction (SAED) patterns respectively corresponded to the (220), (311), (400), (440) and (422) lattice planes of cubic Fe 3 O 4 (Fig. 2e). The Si, Fe and O elements were clearly exhibited in the elemental mapping images (Fig. 2f-i). Particularly, the distribution of Si and Fe elements further confirmed the shell-core structure of the nanochains. The results confirmed that the Fe 3 O 4 nanoparticles could be induced to align in a nanochain though magnetic dipolar interaction under external magnetic field.
Magnetic field distribution around single Fe 3 O 4 nanoparticle and nanochain in the same direction of external magnetic field was analyzed using finite element method (COMSOL Multiphysics), as shown in Fig. 3a-c. It could be seen that the magnetic dipole moment of a Fe 3 O 4 nanoparticle reached a saturated value under the adequately strong magnetic field (Fig. 3a). When the centerlines of two adjacent nanoparticles were aligned with the direction of the external magnetic field, the dipoledipole interaction was attractive (Fig. 3b). In the case of the interaction energy was large enough to overcome thermal fluctuations, the magnetic dipole-dipole force drove the self-assembly of nanoparticles into nanochain along the dipole moment (Fig. 3c). In this condition, the dipole-dipole coupling reached the maximum, and the magnetism of nanochain could be regarded as the magnetic energy coupling among multiple nanoparticles. The magnetic strength of nanochain was higher than that of randomly arranged Fe 3 O 4 nanoparticles.
The magnetic properties of Fe 3 O 4 nanoparticles and Fe 3 O 4 @SiO 2 nanochains were detected and presented in Fig. 3d. It could be seen that both nanoparticles and nanochains exhibited superior magnetism, which was an important advantage for their applicability in biomedicine [22,23]. The saturation magnetization of Fe 3 O 4 was 52.2 emu/g. The relatively low saturation magnetization of Fe 3 O 4 @SiO 2 was due to the introduction of non-magnetic SiO 2 shells decreased the weight ratio of Fe 3 O 4 in Fe 3 O 4 @SiO 2 . This phenomenon was also discovered by M. Tarhini and A. Bitar et al. [24,25].

Physical and chemical properties
The porous scaffold with honeycomb structure was shown in Fig. 4a. The pore size of the scaffold was 800 ± 50 μm, which was proven to be beneficial to cell adhesion and climbing growth [31,32]. The phase composition of the scaffolds was assessed using XRD (Fig. 4b). It can be clearly observed that the diffraction peaks belonged to (010), (110), (203) and (205) planes of PLLA [33]. By contrast, the new diffraction peaks corresponding to (311), (400), (551) and (440) crystal planes confirmed the spinel structure of Fe 3 O 4 in PLLA/Fe 3 O 4 and PLLA/Fe 3 O 4 @SiO 2 scaffolds [34].
The magnetic behaviors of the PLLA, PLLA/Fe 3 O 4 and PLLA/Fe 3 O 4 @SiO 2 scaffolds were shown in Fig. 4c and d. It was clearly seen that the introduction of Fe 3 O 4 and Fe 3 O 4 @SiO 2 endowed the non-magnetic PLLA scaffold favorable magnetic properties. This was conducive to construct magnetic microenvironment in scaffold, which was expected to enhance cell viability and promote cell growth through magnetic stimulation.
Mechanical properties of the scaffolds were evaluated via compressive and tensile tests, with the results shown in Fig. 4e   strength and modulus of the PLLA/Fe 3 O 4 @SiO 2 scaffolds were 41.8 ± 1.6 MPa and 142.6 ± 8.5 MPa, which were increased by 87.4% and 80.6% compared with PLLA scaffolds. Moreover, the PLLA/Fe 3 O 4 @SiO 2 scaffolds also exhibited much higher the tensile strength and strain than those of PLLA and PLLA/Fe 3 O 4 scaffolds, which were 13.34 ± 1.2 MPa and 2.13%, respectively. The high mechanical properties of PLLA/Fe 3 O 4 @ SiO 2 scaffolds were attributed to that the Fe 3 O 4 @SiO 2 nanochains acted as rigid reinforcement to enhance the stress transfer efficiency in the matrix.
The TGA measurement were performed to analyze the thermal decomposition and the corresponding residual weight of scaffolds (Fig. 4g). The slight weight loss below 300 °C of the scaffolds was related to the evaporation of adsorbed water molecules. Obviously, the thermal decomposition temperature of PLLA scaffolds was about 300 ~ 400 °C, while the range of decomposition temperature leftward shifted and narrowed after adding Generally, a scaffold with favorable hydrophilicity is more conducive to cell adhesion [35]. The hydrophilicity of PLLA, PLLA/Fe 3 O 4 and PLLA/Fe 3 O 4 @SiO 2 scaffolds was investigated via water contact angle test. As shown in Fig. 4h  SiO 2 scaffolds exhibited the best hydrophilicity, which was mainly due to the silanol groups of SiO 2 absorbed water molecule via hydrogen bonding.

Cytocompatibility
The cytocompatibility of scaffolds is a necessary and crucial element in the bone repair process because it determines whether cells can adhere, grow, and proliferate on the scaffold [36,37]. Herein, the cytocompatibility of the PLLA, PLLA/Fe 3 O 4 and PLLA/Fe 3 O 4 @SiO 2 scaffolds were assessed. As shown in Fig. 5a, cells adhered well on all scaffolds, indicating that the PLLA, PLLA/Fe 3 O 4 and PLLA/Fe 3 O 4 @SiO 2 scaffolds had good biocompatibility. Particularly, better cell adhesion morphology displayed on PLLA/Fe 3 O 4 @SiO 2 scaffold than that on PLLA and PLLA/Fe 3 O 4 scaffolds at the same time point. Moreover, the cells completely expanded and essentially presented normal topological configuration on PLLA/Fe 3 O 4 @SiO 2 scaffold after 7 days of cultivation, indicating that the Fe 3 O 4 @SiO 2 nanochains in scaffold were more conducive to cell adhesion and expansion.
Cell viability is also an important indicator for evaluating the cytocompatibility of the scaffold [38]. To investigate the cells viability induced by the PLLA, PLLA/Fe 3 O 4 and PLLA/Fe 3 O 4 @SiO 2 scaffolds, the cells were strained with calcein AM. Normally, calcein AM only stains living cells, because calcein AM as a dye can be transformed into a membrane impermeable fluorescent analogue by the cell esterases, and the fluorescence will leak out when the cell membrane is completely damaged [39,40]. As shown in Fig. 5b, the density of living cells in PLLA, PLLA/Fe 3 O 4 and PLLA/Fe 3 O 4 @SiO 2 scaffold groups was significantly enhanced with time, confirming that all scaffolds possessed the ability to enhance cell activity. Notably, the cells increased exponentially from 3 to 7 days with the highest density observing in the cells which cocultured with PLLA/Fe 3 O 4 @SiO 2 scaffold, indicating that the Fe 3 O 4 @SiO 2 nanochains in scaffold significantly enhanced cell viability and promoted cell proliferation.
Cell proliferation is one of the important physiological functions of living cells. To quantitatively study the cell proliferation capacity on the PLLA, PLLA/Fe 3 O 4 and PLLA/Fe 3 O 4 @SiO 2 scaffolds, the CCK-8 assay was carried out (Fig. 5c). It could be clearly seen that the optical density (OD) value of cells on all the scaffolds increased significantly with incubating time. Compared with the PLLA scaffold, higher OD value of cells presented on the PLLA/Fe 3 O 4 scaffold, indicating that the Fe 3 O 4 nanoparticles in the scaffold promoted cell proliferation. Especially, the OD value of cells on PLLA/Fe 3 O 4 @SiO 2 scaffold was markedly higher than that on PLLA/Fe 3 O 4 scaffold, indicating that the Fe 3 O 4 @SiO 2 nanochains further promoted cell proliferation.
As one of the early indicators of osteogenic differentiation [41], ALP activity of cells cultured on the scaffolds for 7 days was qualitatively analyzed (Fig. 6a). It could be clearly seen that the cells cocultured with the PLLA/Fe 3 O 4 @SiO 2 scaffold samples exhibited higher ALP activity than PLLA and PLLA/Fe 3 O 4 samples. As one of the late markers of osteogenic differentiation [42], the Alizarin Red staining was performed to assess the extracellular matrix mineralization of cells cocultured with the scaffolds for 7 days (Fig. 6b). As expected, there were obvious red precipitates in all scaffold groups. It was worth noting that the mineral deposition was significantly enhanced in PLLA/Fe 3 O 4 @ SiO 2 samples compared to other groups, mainly due to the stronger magnetic stimulation effect of Fe 3 O 4 @SiO 2 nanochains. The above results demonstrated that the Fe 3 O 4 @SiO 2 nanochains in scaffold markedly enhanced cell activity and promoted cell proliferation, differentiation, and mineralization.
The bone-related gene expressions including RUNX2, OPN, OCN and OSX on PLLA, PLLA/Fe 3 O 4 and PLLA/ Fe 3 O 4 @SiO 2 scaffolds were investigated (Fig. 7). From an overall perspective, the expression levels of RUNX2, OPN, OCN and OSX on day 7 were greatly higher than on day 3. Especially, the expression level of them on the PLLA/Fe 3 O 4 @SiO 2 scaffold were markedly higher than that on PLLA/Fe 3 O 4 and PLLA scaffolds at any time. The results showed that the Fe 3 O 4 @SiO 2 nanochains provided a more favorable magnetic microenvironment for cell differentiation than Fe 3 O 4 nanoparticles in scaffolds, confirming that the superior capability of Fe 3 O 4 @ SiO 2 nanochains to promote cell differentiation.

Discussion
It is well known that various cells, such as mesenchymal stem cells, osteoblasts, and endothelial cells are magnetically sensitive due to the diamagnetism of cell membranes [43]. Inspired by these, researchers have applied different external magnetic fields to study the roles of magnetic stimulation in bone repair in recent years [44,45]. It was found that the external magnetic fields could induce a series of cell behaviors by regulating cell surface receptors and signaling pathways via magnetic stimulations, thereby accelerating new bone regeneration or inhibiting osteoclast resorption. However, the need of magnetic field generators limits the clinical application of magnetic stimulation to a certain extent. To solve the above problem, it would be an effective means to construct an endogenous magnetic microenvironment in bone scaffolds by introducing magnetic materials. As a highly biocompatible and magnetic materials, Fe 3 O 4 nanoparticles have received clinical approval from the Food and Drug Administration. The scaffolds loaded with Fe 3 O 4 nanoparticles indeed effectively enhanced cell viability and promoted cell proliferation [46,47]. However, the random arrangement of Fe 3 O 4 nanoparticles in the scaffolds greatly compromised their positive magnetic stimulation effects, due to the mutual repulsive between adjacent magnetic dipoles.
In present study, we constructed Fe 3 O 4 @SiO 2 nanochains with uniform shell-core structure by magneticfield-guided interface co-assembly of Fe 3 O 4 nanoparticles (Fig. 2). The simulation analysis results of magnetic field distribution proved the orderly assembly of Fe 3 O 4 nanoparticles in the Fe 3 O 4 @SiO 2 nanochains formed magnetic energy coupling and obtained a highly magnetic micro-field (Fig. 3a-c). The results are consistent with the analysis of Yin Yadong's team [48][49][50]. From the results of magnetic tests, the Fe 3 O 4 @SiO 2 nanochains Fig. 7 The expressions of (a) RUNX2, (b) OPN, (c) OCN and (d) OSX on the scaffolds after 3 and 7 days of culture (n = 5, *p < 0.05, **p < 0.01) still preserved the superior superparamagnetism of Fe 3 O 4 nanoparticles. The good magnetism and high surface areas endowed Fe 3 O 4 @SiO 2 nanochains with great potential for use in biomedicine.
To better understand the biological advantages of Fe 3 O 4 @SiO 2 nanochains in scaffolds, a series of in vitro cell experiments were performed. Compared to PLLA/ Fe 3 O 4 scaffolds, PLLA/Fe 3 O 4 @SiO 2 scaffolds are more conducive to cell adhesion and expansion, especially further enhancing cell viability, proliferation, differentiation, mineralization, and bone-related gene expressions (Figs. 5, 6 and 7). It could be attributed to the stronger magnetic stimulation effect of Fe 3 O 4 @SiO 2 nanochains. In terms of mechanism, the orderly assembly of Fe 3 O 4 nanoparticles obtained magnetic energy coupling, resulting in a highly micro-field that stimulated the surrounding cells to respond (Fig. 8). In this case, the membrane flux of the diamagnetic cell membrane would be modified. Moreover, the strong magnetic singles would activate receptors on the cell membrane, thereby modulating a series of signaling pathways including Ca 2+ channels, mitogen-activated protein kinase (MAPK), bone morphogenetic protein-2 (BMP-2) and integrins [6,10,41,51]. Then, the corresponding downstream transcription factors were regulated, and consequently osteogenesisrelated gene expressions of RUNX2, OPN, OCN and OSX were up-regulated. Hence, the Fe 3 O 4 @SiO 2 nanochains in scaffold possessed great potential in accelerating bone repair.

Conclusions
A magnetic-field-guided interface co-assembly of Fe 3 O 4 nanoparticles had been demonstrated to rationally synthesis unique Fe 3 O 4 @SiO 2 nanochains. The obtained Fe 3 O 4 @SiO 2 nanochains exhibited high magnetic susceptibility and excellent magnetic induction intensity. Importantly, the superior magnetic properties of nanochains enhanced the interaction between PLLA/Fe 3 O 4 @SiO 2 scaffold and cells. As a result, the nanochains in scaffold effectively enhanced cell activity, proliferation, differentiation, and mineralization as well as bone-related gene expressions. These findings